This invention relates to novel serine proteases isolated from a culture medium of the fungus Tritirachium album. The serine proteases of the present invention have a high degree of stability in aqueous solutions and dry detergent formulations. The invention further relates to detergent compositions containing such proteases and to the use of the proteases in detergents and cleaners or spot cleaners. In addition, the present invention further relates to DNA sequences encoding for the proteases and to a method for producing the proteases.
Serine proteases are proteolytic enzymes having a serine residue at their active site. Modification of the active site serine residue by agents such as phenylmethylsulfonylfluoride (PMSF) inactivates these enzymes. There are two classes of serine proteases, chymotrypsin-like proteases and subtilisin-like proteases. Subtilisins are serine proteases which generally act to cleave internal peptide bonds of proteins or peptides. Subtilisins are secreted by a number of Bacillus species and extensively used commercially (see U.S. Pat. No. 3,623,957, J. Miller, 1970, J. Appl. Bacteriol, 33, 207; Ward, O. P. 1983, pp. 251-317, Enzymes and Biotechnology, ed., W. M. Fogarty, Applied Science Publishers, London). Subtilisins have been utilized in a number of detergent formulations (see U.S. Pat. Nos. 1,240,058, 3,749,671; 3,790,482; 4,266,031, U.K. Patent No. 1315937).
The use of proteases in industrial processes which require hydrolysis of protein has been limited due to the enzyme instability under the temperature and pH conditions associated with such processes. Although thermal inactivation of the protease may be the most important factor in restricting the industrial use of a protease, other factors such as lack of effectiveness over broad pH ranges and use of denaturing agents in detergent formulations may also have a detrimental effect regarding the use of proteases in industrial processes. The known Bacillus derived subtilisins are not ideal for all applications as detergent enzymes, in particular, application requiring greater storage stability and activity at broader ranges of pH and temperature. Therefore there is a need for a class of proteases that are characterized by high stability with respect to temperature, pH, denaturing agents and the like.
There also is a need for proteases that are compatible with detergents and have sufficient shelf-life in liquid detergent formulations to be commercially practical. Thermostable fungal serine proteases have been evaluated in detergent applications, including proteases obtained from Tritirachium album (Ebeling, W. et al., 1971, German Offenbach, 1965, 281), Malbranchea pulchella (Ong, P.S. et al., Can. J. Microbiol. 22, 165), Acremonium kiliense, Fusarium and Gibberella spp (Isono, M., et al., 1972, U.S. Pat. No. 3652399). Proteinase K (EC 3, 4, 21, 14) was isolated from Tritirachium album (Ebeling, W. et al., 1974, Eur. J. Biochem. 47, 91-97) and has been extensively studied by different groups (Kraus, E. et al., 1976, Hoppe Seyler's Z. Physiol. Chem. 357, 937-947, ibid. 357, 233-237, and Morihara, K. et al., 1975, Agr. Biol. Chem. 39, 1489-1492). The three dimensional structure of proteinase K is similar to that of subtilisins (Paehler, A. et al., 1983, EMBO J. 3, 1311-1314) and there is about 35% homology of the amino acid sequence of proteinase K with that of subtilisins. (Jany, K-D., et al., FEBS Letters, 199, 139-144). Detergent compatibility of proteinase K has been suggested from its activity in the presence of high concentration of detergents (Hilz, H. et al., 1975, Eur. J., Biochem., 56, 103-108).
Proteolytic enzymes generally catalyze the cleavage of peptide bonds only within a certain range of pH and temperature. Moreover, even under optimal conditions, a proteolytic enzyme retains its activity only when its polypeptide chain is in native conformation. Unfolding of the native structure often occurs when the enzyme is exposed to extremes of pH or temperature or to certain detergent additives such as surfactants and metal-chelating agents. The latter exert their effect especially on enzymes that require metal ions such as Ca.sup.2+ for stabilizing their native structure, i.e., bacterial subtilisin. For proteases, partial unfolding of the native conformation of the enzyme may lead to acceleration of autodigestion and therefore, to irreversible enzyme inactiviation. Because most commercial laundry detergents have an alkaline pH, it is desirable that the enzyme utilized in such detergents be active and stable in a pH range of between 7.5 and 13 and in a temperature range of between 20.degree.-65.degree. C. Moreover, it is desirable that the activity of such enzymes be relatively independent of calcium and magnesium ions and be compatible with surfactants and sequestrant builders. Bacterial serine proteases of the subtilisin family fulfill these requirements to some extent, however their stability in liquid detergent formulation is limited.